The long term goal of this Program is to develop gene transfer methods for the treatment of neural disorders. Three groups that are well integrated have come together to develop methods for using recombinant Adeno-associated virus (rAAV) for the treatment of retinal and CNS neurodegenerative diseases. Project 1 (Muzyczka) proposes genetic experiments to identify the proteins in the substantia nigra and striatum that interact with alpha synuclein. It will specifically examine alpha syn interactions with GRK and PLD2, and develop for the first time somatic knockouts of GRK and PLD2 using AAV vectors. It will also examine the effect of oxidative stress in combination with alpha syn overexpression on neurodegeneration in the substantia nigra. Finally, it will use biochemical techniques to directly identify protein complexes that contain alpha syn. Project 2 (Hauswirth and Lewin) will take the next step toward developing a therapy for P23H rhodopsin RP using the ribozymes they developed in the previous grant period. Further, they will test two new strategies for RP that are likely to be of more general use for all RP diseases. The first is the use of GDNF expression to promote photoreceptor survival. The second is to replace all (wild type and mutant) endogenous rhodopsin mRNAs with a wild type mRNA. If successful, this should prove to be a general approach that could be applied to all genetic RP, regardless of the point mutant involved. Project 4 (Mandel) will extend their preclinical experiments toward developing AAV mediated gene transfer for Parkinson disease. Specifically, they will develop regulatable GDNF constructs that are a prerequisite for clinical applications, do the first comprehensive analysis of the immune response to AAV vectors that are injected into the brain, and test their therapeutic GNDF strategy in a primate model of Parkinson's to obtain dosing information and confirmation of efficacy in a brain model closer to human. Two cores are also proposed. Core A (Administration) will continue in its role of providing fiscal/administrative support, educational programs, and program oversight in the form of internal and external advisors. The Vector Core will continue to improve the efficiency and scaleability of rAAV vectors. In addition to providing the routine service of production and purification of rAAV2-based vectors, the Core will also develop methods for purification of alternative AAV serotypes and capsid mutants to be used in projects 1, 2, and 3.